1. Field of the Invention
The present invention relates to the fields of biochemistry, cellular biology and molecular biology. More particularly, it relates to the field of protein biochemistry, and specifically, to the use of an assay for determining protein folding and solubility.
2. Description of Related Art
There are a wide variety of potential applications for a genetic system enabling rapid and efficient evaluation of protein solubility characteristics in vivo. One of the cornerstones of biotechnology is the ability to express target proteins in functional form in vivo in genetically-engineered organisms. However, many important target proteins are not efficiently expressed in soluble form in bacteria such as E. coli, due at least in part to the complexity of the protein folding process in vivo (Houry et al., 1999). When encountering a target protein that fails to be expressed in soluble form in vivo, the yield of soluble protein can often be improved by optimizing various factors such as the primary sequence of the target protein (Huang et al., 1996) or the genetic background or growth conditions of the bacterium (lung et al., 1998; Brown et al., 1997; Blackwell & Horgan, 1991; Bourot et al., 2000; Sugihara & Baldwin, 1988; Wynn et al., 1992). However, existing assays for protein expression in soluble form are tedious, usually requiring lysis and fractionation of cells followed by protein analysis by SDS-polyacrylamide gel electrophoresis. Using this traditional approach, screening for protein constructs and/or physiological conditions yielding improved solubility is inefficient, and genetic selection is impossible.
Protein folding diseases represent a second area in which protein solubility characteristics are of vital medical and technological importance (Thomas et al., 1995; Dobson, 1999). These diseases, which have proven particularly refractory to pharmaceutical development, are caused either by misfolding of a protein during biosynthesis subsequent to acquiring some mutation (Brown et al., 1997; Thomas et al., 1992; Rao et al., 1994) or by aberrant protein processing leading to the formation of an aggregation-prone product, such as the peptide forming the amyloid plaques associated with Alzheimer's disease (Tan & Pepys, 1994; Harper & Lansbury, 1997), SOD1 in amyotropic lateral sclerosis (Bruijn et al., 1998), α-synuclein in Parkinson's disease (Galvin et al., 1983), amyloid A and P deposits in systemic amyloidosis (Hind et al., 1983), transthyretin fibrils in fatal familial insomnia (Colon & Kelly, 1992) and the intranuclear inclusions associated with polyglutamine expansions which cause Huntington's disease (Martin & Gusella, 1986; HDCRG, 1993; Davies et al., 1997), spinocerebellar ataxia (Wells & Warren, 1998), spinobulbar muscular atrophy (La Spada et al., 1991), and Machado-Joseph Disease (Kawaguchi et al., 1994). The ability to rapidly and efficiently screen for protein solubility in vivo could also be applied to the development of assays for pharmaceutical compounds preventing the misfolding or aggregation of proteins involved in protein folding diseases (i.e., assays for compounds that prevent precipitation of such aggregation-prone proteins).
Thus, there remains a need in the field for improved methods of screening for protein folding and solubility.